This long-term goal of this project is to increase understanding of the modulation of fibrinolysis by serum proteins thereby providing information vital for improved diagnosis and therapy of thrombotic cardiovascular disorders. The recent discovery that the histidine-rich glycoprotein (HRG) of serum interacts strongly with plasmin and its zymogen, plasminogen, has opened a new avenue for study of the homeostatic balance between blood coagulation and clot lysis. Preliminary evidence shows that HRG not only affects the extent of activation of plasminogen to plasmin but also inhibits the lytic activity of plasmin and decreases the inhibitory action of Alpha2-antiplasmin. Thus, HRG is intimately connected with every facet of fibrinolysis. The questions we address include: What are the levels of HRG in the serum of patients with fibrinolytic disorders or undergoing thrombolytic therapy? What are the mechanisms of the effects of HRG on the activation, enzymatic activity and inhibition of plasmin(ogen)? What is the physico-chemical nature of the HRG-plasmin(ogen) interaction? What are the concentrations of the HRG-plasmin(ogen) complex in vivo? What factors (e.g. heparin, calcium and metals) alter the interrelations of HRG with plasmin(ogen)? We propose a multi-faceted research program with four major portions to determine the physiological consequences and mechanism of the interaction of HRG with plasmin(ogen). First, the levels of HRG will be measured in human subjects with fibrinolytic, thrombotic and other cardiovascular conditions. Second, the effects of the interaction fo HRG on the activation of plasminogen, the activity of plasmin and the inhibition of plasmin by serum protease inhibitors will be assessed using both synthetic chromogenic peptide substrates and the natural substrate, fibrin. Third, the affinity and kinetics of the interaction and the structural features of the two proteins important to their functional interplay will be determined. Fourth, the HRG-plasminogen complex in vivo will be measured using unique immunoadsorbent techniques currently being developed in this laboratory. In each area, the consequences of substances known to be effectors of plasmin or to be bound by HRG will be assessed. In the future, we plan to extend this work to the metabolism of HRG and to HRG-tissue interactions particularly with tissues having high levels of plasminogen activator activity.